The Na-K-2Cl (sodium-potassium-2-chloride) cotransporter is an electroneutral membrane protein that plays several important roles in cellular physiology. Recent data suggest that the Na-K-2CI cotransporter may play a role in airway smooth muscle relaxation. However, molecular identification of this co-transporter protein airway smooth muscle has not been confirmed. More fundamentally, this cotransporter protein in airway smooth muscle has not been confirmed. More fundamentally, this cotransporter regulates salt and water secretion across epithelial cells and maintenance and regulation of cell volume and ion gradients in epithelial and non-epithelial cells (e.g., erythrocytes). The erythrocyte cotransporter appears important because the level of activity in adults with essential hypertension has been correlated with the diuretic and natriuretic responses to furosemide, suggesting genetic variation in cotransporter regulation. Characterization of the Na-K-2Cl cotransporter in human erythroid progenitor cells and neonatal erythrocytes would provide information about developmental changes in the cotransporter expression and function. The present proposal focuses on the Na-K-2CL cotransporter in the neonatal guinea pig and human fetal airways and in the human neonatal erythrocyte and its precursor, the erythroid progenitor cell. The following hypotheses will be tested: 1) the Na-K-2Cl cotransporter is expressed in airway smooth muscle; 2) changes in erythrocyte cotransporter expression and activity occur with maturation; and 3) the regulation of the human Na-K-2Cl cotransporter is similar to that found in animal paradigms. Results of this project will further our understanding of the importance of the Na-K-2Cl cotransporter by providing evidence for its role in loop diuretic mediated airway smooth muscle relaxation. Also, characterization of the Na-K-2Cl cotransporter in the human erythrocyte with the study of its function and changes in maturation may provide new insights into the developmental regulation of this cotransporter as well as in understanding kidney function and the development of essential hypertension.